A preclinical therapy model for childhood neuroectodermal tumours

Childhood cancers show fundamental differences to most common adult solid tumours in their cancer-causing genetics, cell biology and their local tissue microenvironment. Effective treatments will be attainable when the molecular events that are specific to childhood tumourigenesis are better understood. However, it is in this context critical to consider both species and developmental aspects when looking for the relevant signalling. An influence from the microenvironment on clonal dominance is likely contributing to the disparity between primary and metastatic tumours seen in many patients, as well as inter-tumour heterogeneity between patients with the same tumour type. In this thesis a novel concept is presented for preclinical studies of embryonic tumours in a recently described humanised model. Benign human experimental teratoma was generated in NOD SCID gamma (NSG) mice from diploid bona fide pluripotent stem cells (the PSCT model). An abundant presence of developing early neural components suggests this human embryonic model to be uniquely suited for in vivo transplantation studies of primitive neuroectodermal tumours originating early in life. Following transplantation of tumour cell lines (Paper I), or patient biopsy material (Paper II) the initial microcolonisation showed a tumour type specific tissue tropism in that growth was instigated into specific embryonic tissue compartments, constituting the preferred cellular context/niche supporting initial colonisation and expansion. The phenomenon of microcolonisation reflects in this context an ability of tumour cell clones to comply or adapt to new environments, a feature which has great impact on metastasis and clinical prognosis. Chemotherapy is widely accepted as part of first-line therapy for high-risk paediatric neuroblastoma. In a third study (Paper III) we have examined chemotherapy responsiveness/resistance of neuroblastoma in the PSCT model. Two well-characterized neuroblastoma cell lines were subjected to chemotherapy using the anthracycline drug doxorubicin and we report for the first time in situ studies performed in a human homologous embryonic in vivo microenvironment. Here we combined classical histopathology with high throughput single cell screening for preclinical drug evaluation. Using laser scanning cytometry for screening of chemotherapy-induced changes of nuclear DNA index (sub G1 fraction), in combination with immunohistochemistry and cytology for proliferation, apoptosis and cell death, we could demonstrate dose dependent and diversified responses to single-dose regimens of doxorubicin. Following a recurrent doxorubicin regimen (repeated administration with 48h interval), we observed the presence of an asynchronous response within individual tumour colonies, with cells exhibiting cytotoxic effects or enhanced proliferative index. The results illustrate the feasibility of the approach and are encouraging for clinically relevant studies of patient material regarding intra tumour heterogeneity and asynchronous response to therapy. Furthermore, the findings demonstrate several important advantages using the PSCT model compared to employing conventional preclinical in vivo models and lead us to propose that the use of this human embryonic microenvironment is a well-needed complement for preclinical in vivo studies of primitive neuroectodermal tumours originating early in life

Stem Cell Therapy in Injured Vocal Folds : A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (P <= 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (P <= 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives

Stem Cell Therapy in Injured Vocal Folds : A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (P <= 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (P <= 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives

Stem Cell Therapy in Injured Vocal Folds : A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (P <= 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (P <= 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives

Алгоритмизация и программирование

Представлены задания к лабораторным работам, с помощью которых выполняется разработка и практическая реализация основных видов алгоритмов на языке высокого уровня Оbject Pascal. Рассмотрены сложные структуры данных и алгоритмы их обработки, а также алгоритмы вычислительной математики. Для студентов специальности 1-53 01 01 «Автоматизация технологических процессов и производств (по направлениям)» дневной формы обучения

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation

The effects of doxo on PSCT non-malignant embryonic tissues.

<p>Immunohistochemistry staining of formalin-fixed paraffin-embedded PSCT histological slides, following intra peritoneal injection of the host mouse with 8mg/kg doxo (A-D), or 4+4mg/kg doxo (E-H). High frequencies of positive staining for Ki67 in tissues compatible with neural epithelium, muscle and cartilage can be seen, indicative of extensive proliferation (A,C,E,G). Low frequencies of positive staining for cleaved caspase 3 can be seen in NE, muscle and cartilage, indicative of low frequencies of apoptosis (B,D,F,H). Size bars: 50μm.</p

Micro colonisation of IMR-32 tumours in the PSCT model.

<p>(A) Schematic illustration; NB cells were injected into an arbitrary position centrally in the PSCT cellular mass, resulting in multiple micro-colonisations from migrating NB cells. (B) A representative FFPE section of a PSCT with four IMR-32 colonies indicated (red borders). (C) IMR-32 colony surrounded by loose mesenchyme. LM = loose mesenchyme; NE = neural epithelium; C = cartilage; M = muscle; Blue arrows = vessels. Size bars: B:5mm, C:500μm.</p

Data set for IMR-32 <i>in vitro</i>.

<p>Data set for IMR-32 <i>in vitro</i>.</p

Data set for BE(2)-C <i>in vitro</i>.

<p>Data set for BE(2)-C <i>in vitro</i>.</p